Title of Invention	A METHOD FOR PEPTIDE SYNTHESIS, A COMPOSITION OF PEPTIDE FRAGMENTS AND PEPTIDES MADE THEREBY
Abstract	The present invention relates to a method for synthesizing a peptides of peptides referred to as T-1249 and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides on interest (e.g., T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce full-length T-1249 and T-1249-like peptides. ABSTRACT IN/PCT/200200154/CHE A method for peptide synthesis, a composition of peptide fragments and peptides made thereby The present invention relates to a method for synthesizing a peptide of peptides referred to as T-1249 and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides on interest (e.g., T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce ful-length T-1249 and T-1249-like peptides.

Title of Invention

A METHOD FOR PEPTIDE SYNTHESIS, A COMPOSITION OF PEPTIDE FRAGMENTS AND PEPTIDES MADE THEREBY

Abstract

The present invention relates to a method for synthesizing a peptides of peptides referred to as T-1249 and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides on interest (e.g., T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce full-length T-1249 and T-1249-like peptides. ABSTRACT IN/PCT/200200154/CHE A method for peptide synthesis, a composition of peptide fragments and peptides made thereby The present invention relates to a method for synthesizing a peptide of peptides referred to as T-1249 and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides on interest (e.g., T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce ful-length T-1249 and T-1249-like peptides.

Full Text	The present invention relates, first, to methods for the synthesis of peptides, in particular peptides referred to herein as T-1249 (SEQ ID N0:1) and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which can act as intermediates in the synthesis of the peptides of interest (e.g.. T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce full-length T-1249 and T-1249-like peptides. The present invention still further relates to methods for the purification of peptides, in particular T-1249 and T-1249-like peptides, and the individual peptide fragments which act as intermediates in the synthesis of the subject peptides. 2. BACKGROUND Recently, a large number of peptides have been identified which exhibit an ability to inhibit fusion-associated events, and, importantly, also exhibit potent antiviral activity. See, for exan^le, U.S. Patent Nos. 5,464,933; 5,656,480; PCT Publication Nos. WO 94/28920; WO 96/19495. As these peptides come to be used extensively, for example as therapeutics, the need arises for an ability to synthesize them in large scale quantities. While techniques exist for peptide synthesis, (see, e.g.. Mergler et al., 1988, Tetrahedron Letters 29:4005-4008; Mergler et al., 1988, Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), "Peptides, Chemistry and Biology, ESCOM, Leiden, 1992, 525-526; and Riniker et al., 1993, Tetrahedron Letters 49:9307-9320) no techniques currently exist which can be utilized for large scale, economical production of easily purified peptides such as T-1249. 3. SUMMARY OF THE INVENTION The present invention relates, first, to methods for the synthesis of peptides, in particular peptides referred to herein as T-1249 (SEQ ID NO:l) and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. Generally, the methods of the invention comprise synthesizing specific side-chain protected peptide fragment intermediates of T-1249 or a T-1249-like peptide on a solid support, coupling the protected fragments in solution to form a protected T-1249 or T-1249-like peptide, followed by deprotection of the side chains tc yield the final T-1249 or T-1249-like peptide. A preferred embodiment of the methods of the inver.tion involves the synthesis of a T-1249 peptide having an ami'.no acid sequence as depicted in SHQ ID NO:l. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides of interest (e.g., T-1249) . The peptide fragments of the invention include, but are not limited to, those having amino acid sequences as depicted in Table 1 below: The present invertuion still further relates to particular groups of peptide fragments which act as intermediates in the synthesis of the peptide of interest. The groups of peptide fragments according to the invention include Groups 1-6, as designated in Table 2 below. This invention is based, in part, on the inventors' unexpected discovery that certain, combinations of solid phase liquid phase methetic reactiorns allow high p-urity T-1249 and T-1249-like peptides to be manufactured for the first time on a large scale with high throughput and high yield. In particular, in accordance with the methods of the invention, T-1249 and T-l249-like peptides may be synthesized on a scale of one or more kilograms. It has been found that by selecting the specific T'1249 peptide fragments of the invention for solid phase synthesis, the highly efficient coupling of solid phase techniques may be exploited without having to use the 3-, 4- or even 5-fold excess of amino acids and reagents that are normally required in solid phase synthesis. The methods of the invention use only about an O.S-fold excess (about 1.5 equivalents) of amino acid in the solid phase synthesis of the peptide fragments of the invention. This reduction in the amount of amino acid and reagents makes the methods of the invention suitable for large scale synthesis of T-1249 and T-1249-like peptides. In addition, the inventors have surprisingly foxind that certain peptide fragments may be synthesized in the solid phase at a loading of about >o.5 mmol per gram of solid phase resin. This loading significantly enhances throughput over the loading range of 0.25 to 0.35 mmol per gram of resin typically achieved in solid phase peptide synthesis. Moreover, the inventors have found that synthesizing selected peptide fragments in the solid phase using super acid sensitive resin produces peptide fragments of unusually high purity. Chromatographic techniques are not necessary to purify the peptide fragments produced according to the invention; the fragments are simply put through precipitacion and/or trituration steps before use, or used as obtained directly from the resin. Use of a super acid sensitive resin allows the synthesized, protected peptides of the invention to be cleaved from the resin without concomitant removal of the side-chain protecting groups. This reduces impurities, and allows peptides comprising 10 amino acids or greater to be synthesized in high purity and yield. The impurity profile of T-1249 and T-1249-like peptides which are synthesized in the solution phase according to the methods of the invention by coupling of the high purity peptide fragments produced according to the invention consists of fragments that did not couple, and contains significantly lower levels of closely related deletion analogues than T-1249 and T-1249-like peptides synthesized according to conventional techniiiues, e.g. . solely solid phase synthesis. Accordingly, T-1249 and T-1249-like peptides produced according to the invention are much easier to purify than those produced according to conventional techniques. In particular, T-1249 produced according to the methods of the present invention may be easily purified to >90% purity in single pass chromatography. For example, in accordance with the methods of the invention, T-1249'can be purified in amounts of 400g or more using a 5 inch column. In contrast, T-1249 prepared using conventional solid phase synthesis The Examples presented in Section 9 below, demonstrate such combinatorial syntheses of T-1249 full-length peptides. The T-1249 and T-1249-like peptides and intermediates may be produced on a scale of one or more kilograms by the methods of the invention. The presenc inventors have also unexpectedly found that peptides such as T-1249 and other T-l249-like peptides, as well as certain peptide fragments described herein may be purified using high capacity materials because of the high purity of T-1249 after solution phase synthesis. Thus, the present invention still further relates to methods for the purification of peptides, in particular T-1249 and T-1249-like peptides, and the individual peptide fragments which act as intermediates in the synthesis of the subject peptides. 3.1 DEFINITIONS The amino acid notations used herein are conventional - and are as follows: 4. BRIEF DESCRIPTION OP THE FIGURES FIG. 1: T-1249 three fragment approach. This figure depicts the scheme followed in the Example presented in Sections 8 and 9 below, for the synthesis of full-length T-1249 beginning with intermediate peptide fragment Group 1, as shown in Table 2, above, and depicts one, non-limiting embodiment of the methods of the invention. 5. DETAILED DESCRIPTION OF THE IHVEHTION 5.1 FULL-LENGTH PEPTIPES The present invention relates to methods, peptide fragments, groups of peptide fragments which can be used to synthesize the peptide known as T-1249. T-1249 is a 39 amino acid residue polypeptide whose sequence is derived from HIV-1, HIV-2 and SIV gp41 viral polypeptide sequences. T-1249 has the following amino acid sequence; NH2-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-COOH It will be understood that the methods, fragments and groups of fragments and techniques utilized for choosing the fragments and groups of fragments of the present invention may be used to synthesize T-1249-like fragments in addition to T-1249. The term "T-1249-like" as used herein means any HIV or non-HlV peptide listed in International Publication No. PCT/US99/11212, filed May 20, 1999, which is hereby incorporated by reference in its entirety. In addition to T'1249 and the T-1249-like peptides described above, the methods, fragments and groups of fragments of the present invention may be used to synthesize peptides having modified amino and/or carboxyl terminal ends. Taking T-1249 as an example, such peptides can be of the formula: X-WQEWEQKITALLEQAQIQQEKNEyELQKLDKWASLWEWF-Z wherein X represents an amino group; a hydrophobic group selected from the group consisting of carbobenzoxyl, dansyl, and T-butyloxycarbonyl; an acetyl group; a 9-fluoroenyl-methoxy-carbonyl (FMOC) group; or a macromolecular carrier group selected from the group consisting of lipid-fatty acid conjugates, polyethylene glycol, and carbohydrates; and Z represents a carboxyl group; an amido group; a T-butyloxycarbonyl group; a para-nitrobenzyl ester group; or a macromolecular carrier group selected from the group consisting of lipid-fatty acid conjugates, polyethylene glycol, and carbohydrates. In a preferred embodiment, the methods of the invention are used to synthesize the peptide having the above formula wherein X is an acetyl group and Z is an amide group. Techniques for addition of such "X" and "Z" groups are well known to those of skill in the art. In a preferred method, T-1249 and T-1249-like peptides and intermediates can be purified using any non-silica based column packing (for maximization of loading capacity) including but not limited to zirconium-based packings, poly¬styrene, poly-acrylic or other polymer based packings which are stable at high and low pH ranges. For example, among the non-silica-laded column packing exhibiting a broad pH range that includes pH values greater than seven are ones sold by Tosohaas (Montgotneryville, PA) , Columns packed with such material can be run in low, medium or high pressure chromatography according to standard techniques well known in the art. The Examples presented in Section 9, below, demonstrate the successful synthesis of T-1249 peptides via coupling of peptide intermediates described, below, in Section 5.2. 5.2 PEPTIDE IHTERMEDIATES The present invention encompasses, but is not limited to, peptide fragment intermediates of T-1249 and T-l249-like peptides with specific amino acid sequences as listed in Table 1 above, and the groups of peptide fragment intermediates listed in Table 2. Such peptide intermediates, especially in groups as listed in Table 2, below, can be utilized to produce T-1249 and T-1249 like peptides. Any one or more of the side-chains of the amino acid residues of peptide fragments listed in Table 1 or 2 may be protected with standard protecting groups such as t-butyl (t-Bu), trityl (trt) and t-butyloxycarbonyl (Boc) . The t-Bu group is the preferred side-chain protecting group for amino acid residues Tyr(Y), Thr{T), Ser(S), Glu(E) and Asp (D) ; the trt group is the preferred side-chain protecting group for amino acid residues, Gln(Q) and Asn(N); and the Boc group is the preferred side-chain protecting group for amino acid residues Lys(K) and Trp(W). Preferably, the Gln(Q) residues of the peptide fragments of the invention are protected with trityl (trt) groups. However, if lower solubility of any of the peptide fragments of the invention in organic solvents is desired, the trityl protecting groups may be eliminated from any one or more of the glutamine residues of the fragments. Preferably, the Asn(N) residues of each peptide fragment of the invention are protected. In addition, it is preferred that the Trp(W) residues are protected with a Boc group. Protected peptide fragments according to peptide formulas 1-5 listed in Table 1 above include, but are not limited to, the compounds listed in Table 3 below. Any one or more of the side-chains of the amino acid residues of the peptides listed in Table 3 above may be protected with standard side-chain protecting groups such as tBu, trt and Boc, as described above. Representative synthesis of peptides from Table 3 are presented in Sections 7 and 8 below, which utilize the general techniques discussed in Section 5.3, below. 5.3 PEPTIDE SYNTHESIS As discussed above, some of the individual peptide fragments of the invention are preferably made using solid phase synthesis techniques, while other peptides of the invention are preferably made using a combination of solid phase and solution phase synthesis techniques, said syntheses culminating in the production of T-1249 or T-1249-like peptides as described herein. However, it will be understood that the peptide fragments of the invention/may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, which is incorporated herein by reference in its entirety. The peptides of the invention may alternatively be synthesized such that one or more of the bonds which link the amino acid residues of the peptides are non-peptide bonds. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds, to name but a few. In yet another embodiment of the invention, T-1249 and T-1249-like peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the stability, reactivity and/or solubility of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxyl, dansyl, acetyl or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides' amino termini. (See "X" modification of T-1249, described above.) Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini. Similarly, a para-nitrobenzyl ester or benzyl ester group may be placed at the peptides' carboxy termini. (See "Z" modification of T-1249, described above.) Technic[ues for introducing such modifications are well known to those of skill in the art. Further, T-1249 and T-1249-like peptide(s may be synthesized such that their steric configuration as altered. For example, the D-isomer of one or moreof the amino acid residues of the peptide may be used, rather than the usual L-isomer. Still further, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well known non-naturally occuring amino acid residues. Alterations such as these may serve to increase the ^ stability, reactivity and/or solubility of the peptides of the invention. Any of the T-1249 or T-1249-like peptides may be synthesized to additionally have a macromolecular carrier group covalently attached to its amino and/or carboxy termini. Such macromolecular carrier groups may include, for example, lipid-fatty acid conjugates, polyethylene glycol, carbohydrates or additional peptides. The "X" modification of T-1249 described above may therefore additionally represent any of the above macromolecular carrier groups covalently attached to the amino terminus of a peptide, with an additional peptide group being preferred. Likewise, the "Z" modification of T-1249 described above may additionally represent any of the macromolecular carrier groups described above. Preferably, the peptide fragments of the present invention are synthesized by solid phase peptide synthesis (SPPS) techniques using standard FMOC protocols. See, e.O, . Carpino et al.. 1970, J. Am. Chem. Soc. 92{19}:5748-5749; Carpino et al.. 1972, J. Org. Chem. 37 (22):3404-3409. In a preferred embodiment, the solid phaae synthesis of the peptide fragments of the present invention is carried out on super acid sensitive solid supports which include, but are not limited to, 2-chlorocritYl chloride resin (see, e.g.. Barlos et al,. 1989, Tetrahedron Letters 30(30):3943-3946} and 4-hydroxymethyl-3-methoxyphenoxybutyric acid resin (see, e.g.. Seiber, 1987, Tetrahedron Letters 28(49):6147-fii50, and Richter et al.. 1994, Tetrahedron Letters 35(27^:4705-4706). Both the 2-chlorotrityl chloride and 4-hydroxymethyl-3-methoxyphenoxy butyric acid resins may be purchased from Calbicchem-Novabiochem Corp., San Diego, CA. General, non-limiting procedures for production and loading of resins which can be utilized in solid phase peptide synthesis are described herein. In addition, the examples present in Section 6, below, describe exemplary resin preparations. Resin loading can be performed, for example, via the following techniques: The resin, preferably a super acid sensitive resin such as 2-chloiotrityl resin, is charged to the reaction chamber. The resin is washed with a chlorinated solvent such as dichloromethane (DCM). The bed is drained and a solution of 0.5 - 1,5 equiifalents of an amino acid with an 0.2 to 0.5 excess of diisopropylethylamine (DIEA) in about 8-10 volumes of dichloroethane (DCE) is added. The N-terminus of the amino acid should be protected, preferably with Pmoc, and the side chain of the amino acid should be protected where necessary or appropriate. The mixture is agitated with nitrogen bubbling for 2-24 hours. It should be noted that a chlorinated solvent such as DCM or DCE is desired for adequate swelling of the 2-chlorotrityl resin. After agitation, the bed is drained and washed with DCM. The active sites on the resin are endcapped with a 9:1 MeOH:DIEA solution for about 20-30 minutes. The bed is drained, washed four times with DCM and dried with a nitrogen purge to give the loaded resin. Fmoc is the preferred protecting group for the N-terminus of the amino acid. Depending on which amino acid is being loaded, its side chain may or may not be protected. For example, when tryptophan (Trp) is loaded, its side chain should be protected with Boc. However, it is not necessary to protect the side-chain of leucine (Leu). Preferably, glutamic acid (Glu), aspartic acid (Asp), threonine (Thr) and serine (Ser) are protected as t-butyl ethers or t-butyl esters, and tryptophan (Trp) and lysine (Lys) are protected as t-butoxycarbonyl carbamates (Boc). The amide side-chain of asparagine (Asn) and glutamine (Gin) may or may not be protected with trityl groups. The Fmoc-protected amino acids used in loading the resin and in peptide synthesis are available, with or without side-chain protecting groups as required, from multiple vendors, including Senn or Genzyme. As an alternative to the above procedure, the resin may be purchased already loaded with the appropriate amino acid. The Examples presented in Section S, below, describe exemplary resin preparations. Solid phase peptide synthesis techniques can be performed as, for example, according to the following, non-limiting techniques: The loaded resin is added to the reaction chamber and conditioned with a solvent, preferably methylene chloride (DCM; at preferably about 10 vol.) with nitrogen agitation or stirring for about 15 minutes to swell the resin beads. DCM is required for adequate swelling of the 2-chlorotrityl resin. The resin volume will increase 3-6 fold in the reaction chamber as the beads swell and the active sites vinfold and become accessible to reaction. After tHe resin is swelled, the solvent is drained from the reaction chamber. Removal of the Fmoc (9-fluroenyl-methyloxycarbonyl) protecting group from the terminal amine or the resin can be accomplished by treating the resin with 2 aliquots of a 20% solution of piperidine in N-methyl-2-pyrrolidinone (NMP) for about ten minutes each. The volume of the 20% solution of piperidine in NMP required for each aliquot will depend on the scale of the reaction being run. The resin is then washed 5-7 times with aliquots of NMP (aboutio vol.) to remove the Fmoc by-products (i.e., dibenzofulvene and its piperidine adduct) and residual piperidine. A chloranil test may be used to determine if the removal of residual pyridine is complete. The chloranil test solution is prepared by adding a drop of a saturated solution of chloranil in toluene to about 1 mL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that residual piperidine is still present. The NMP washing is repeated until the blue or violet color is no longer observed. Meanwhile, the subsequent amino acid in the sequence to be added to the resin is activated for reaction at its carboxy terminus. The amine terminus of each amino acid should be protected with Fmoc. Depending on which amino acid is being added, its side chain may or may not be protected. Preferably, the side-chains of tyr(y), Thr(T), Ser(S), Glu{E) and Asp(P) are protected with t-Bu, the side-chains of Gln(Q) and Asn(N) are protected with trt, and the side-chains of Lys(K) and Trp{w) are protected with Boc. It is not necessary for the side-chains of Leu or l1e to be protected. The amino acid can be activated as follows. The Fmoc-protected amino acid (1.5 eq) , 1-hydroxybenzotriazole hydrate (HOBT) (1.5 eq) , and diisopropyl-ethylamine (DIEA) (1.5 eq) are dissolved in a polar, aprotic solvent such as N-methyl pyrrolidinone (NMP), dimethyl formamide (DMF) or dimethyl acetamide (DMAC) (about 7.5 vol.) at room tenperatiire. The solution is chilled to O-S'C, and then O-benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or O-benzotriazol-l-yl-tetramethyltetrafluoroborate (TBTO)(1.5 eq) is added followed by stirring for 5-15 minutes to dissolve. It is important that activation is carried out at O-B'C to minimize racemization of the amino acid. The HBTU is the last reagent added to the cold solution since activation and racemization cannot take place in its absence. The solution of activated amino acid is charged to the drained resin, washing in with DCM (approximately 2.5 vol). Note that activation of the amino acid is carried out in NMP due to the insolubility of HBTU in DCM. However, DCM is added to the reaction at this point to maintain adequate swelling of the resin beads. The reaction is agitated with Nj bubbling for about 1 hour at 20-30°C. Coupling completion may be monitored with a qualitative ninhydrin test as described below. To check for completion of the reaction using the qualitative ninhydrin test, a 2-20 mg sample of the resin can be withdrawn and washed clean with methanol. To the sample is added 3 drops of a 76% solution of phenol in ethanol, 4 or 5 drops of a 0.2 mM KCN solution in pyridine, and 3 drops of a 0.28 M solution of ninhydrin in ethanol. The sample is diluted with ethanol to a volume of about 0.5 mL and placed in a heat block at about 75°C for 5-10 minutes- A blue or violet color is a positive indication for the presence of free amines, indicating that the reaction is not yet complete. The sample can be diluted further to a volume of about 3 mL to more easily gauge the degree of color change in the concentrated sample. If a positive ninhydrin test is observed after one hour, the coupling reaction is continued for an additional hour. If the positive ninhydrin test persists after 3 hours, the resin is drained, washed one time in approximately 10 volumes of NMP, and the coupling reaction is repeated using 0.5-1 equivalent of activated amino acid. If the resin is to be stored overnight between coupling cycles, the resin bed may be drained and covered with NMP under a nitrogen blanket. Alternatively, the bed may be drained, stored under a nitrogen blanket, then conditioned with a DCM wash prior to proceeding with the next coupling cycle. If the con^leted fragment is to be stored overnight prior to cleavage, the resin bed should be washed free of NMP with IPA because significant Fmoc deprotection can occur in NMP. After the coupling is judged complete, the resin is drained and washed with 3 aliquots (approximately 10 vol.) of NMP. The cycle is repeated for subseguent mers (i.e., amino acids) of the peptide fragment. Following the final coupling reaction, the resin is washed with 4 aliquots (about 10 vol.) of NMP, then with 2 aliquots (approximately 10 vol.) of DCM and 2 IPA. The resin-bound peptide may be dried with a nitrogen purge or in an oven. Peptides sj-nthesized via solid phase synthesis techniques can be cleaved and isolated according to, for example, the following non-limiting techniques: The peptide may be cleaved from the resin using techniques well known to those skilled in Che art. For example, solutions of 1% or 2% trifluoroacetic acid (TFA) in DCM or a combination of a 1% and a 2% solution of TFA in DCM may be used tc cleave the peptide. Acetic acid (HOAC), hydrochloric acid (HCl) or formic acid may also be used to cleave the peptide. The specific cleavage reagent, solvents and time required for cleavage will depend on the particular peptide being cleaved. After cleavage the cleavage fractions are subjected to standard work-up procedures to isolate the peptide. Typically, the combined cleavage fractions are concentrated under vacuum, followed by reconstitution with polar aprotic or polar aprotic solvents such as ethanol (EtOH), methanol (MeOH), isopropyl alcohol (IPA), acetone, acetonitrile (ACN) , dimethyl formamide (DMF), NMD, DMAC, DCM, etc., followed by precipitation or crystallization with antisolvent such as water or hexanes, and collection by vacuum filtration. Alternatively, the product may be triturated with organic solvents or water after isolation of the peptide. Accordingly the present invention provides a method for synthesizing a peptide of the formula: X- WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO: 1), comprising: (a) reacting a side-chain protected peptide of the formula: EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:6), wherein the amino termninus is deprotected; with a side-chain protected peptide of the formula: X-WQE\VEQKITALL-COOH (SEQ ID N0:2) in a known manner, to yield a side-chain protected peptide of the formula: X-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:1); wherein X is a protecting group, an acetyl group or a macromolccular carrier group; and wherein Z is a protecting group, or a macromolecular carrier group. The Examples presented in Sections 7.1 - 7.6 below, present solid phase syntheses of peptide intennediates as shown in Tables 1, 2 and/or 3. For synthesis of full length T-1249 peptides, the peptide intermediates of Table 1, above, can be coupled together to - yield the T-1249 peptide. For example, the groups of peptide intermediates listed in Table 2, above, can be coupled together to produce T-1249 full-length peptide. Representative examples of the synthesis of full-length T-1249 from intermediate peptide fragments are presented in Section 9, below, and are depicted schematically in Figure 1. In certain embodiments, a three fragment approach for synthesis of T-1249 can be followed. A "three fragment approach" synthesis refers to a T-1249 synthesis scheme which begins with three T-1249 intermediate peptide fragments that are synthesized and coupled using solid and liquid phase synthesis techniques into a full-length T-1249 peptide. Intermediate peptide fragment groups 1, 2, 3 and 4 shown in Table 2, above, represent preferred groups. Figure 1 depicts an exemplary three fragment approach which utilizes Table 2 peptide intermediate Group 1 to synthesize full-length T-1249. For this group, it is noted that amino acid residue 39 (the T-1249 carboxy 1-terminal amino acid residue) is introduced individually during the fragment coupling process. The culmination of the T-1249 synthesis scheme shown in Figure 1 is demonstrated in the example presented in Section 9.1. Solution phase peptide synthesis techniques well known to those of skill in the art may be utilized for synthesis of the peptide intermediate fragments of the invention. The Examples presented in Section S describe exemplary solution phase peptide synthesis of peptide intermediates listed in Tables 1, 2, and/or 3. For example, among the non-silica-laded column packing exhibiting a broad pH range that includes pH stability values at high or low pH are sold by Tosohaas (Montgomeryville, PA). The air sensitive 2-chlorotritYlchloride resin (Senn Chemicals, Lot A 3573, 1 eq, 12.0 inmol, 10.0 g) is added to a 250 mL round bottom flask and immediately treated with a prepared solution of FmocLeuOH (1.0 eq. , 12 mmol, 4.24 g) and Diisopropyl ethyl amine (5eq., 60.0 mmol, 5.20 mL) in DCN {10 vol, 100 mL) The slurry is capped and stirred for 3 hours. The solvent is removed by filtration and the resin is v.'ashed with DCM (5 vol, 50 mL) . The remaining active sites on the resin are end-capped by treating resin with 9:1 MeOH:DiEA (5 vol, 5.0 mL DJEA and 45 mL MeOH) for 30 minutes. The solvent is removed and the resin is washed with 3x5 volumes of DCM. The resin is dried to constant weight affording 13.2 g of loaded resin with a calculated loading of 0.98 mmol FmocLeuOH per gram. This Method can also be used for Loading FmocTrp (Boc)OH onto 2-CTC resin. Described herein, in Sections 6.2-6.3, are examples in which chlorotrityl chloride resins were substituted with amino acids which can be utilized in conjunction with solid phase synthesis of the peptides and peptide intermediates-described herein. All of the peptides and peptide fragments of the present invention may be synthesized by solid phase peptide synthesis (EPPS) using the loading procedures described in Sections 6.2 and 6.3 below. Procedure: The 2-chlorotrityl chloride resin (25 g, 1 eq.) was charged to a 500 mL peptide chamber and washed with 250 mL of DCM. The bed was drained and a solution of the Fmoc-Trp(Boc)-OH {1.5 eq) and the DIEA (1.7 eq) in 10 volumes of DCE was added. The mixture was agitated with nitrogen bubbling for 2 hrs. The bed was drained and washed with 250 mL DCM- The active sites on the resin were end-capped with 200 mL of a 9:i MeOHiDIEA solution for 20 minutes. The bed was drained, v/ashed with 4 x 250 rnL of DCM, and dried with a nitrogen purge to give 34.3 g of loaded resin. Quantitative HPLC analysis was performed by cleaving the Fmoc-amino acid from the resin and assaying versus a standard. HPLC assay of the material showed a loading of the resin at 0.68 mmol/g. Column: Phenomenox Jupiter C18; 300A; 5μ Plow rate: 1 mL/min Detection: UV at 260 nm Mobile phase: A: 0.1% aqueous TFA B: 0.1% TFA in acetonitrile 65% B isocratic Retention time: Approximately 14 minutes Procedure{ The resin was charged to a 3 L peptide chamber and washed with 1.5 DCM. The bed was drained and a solution of the FmocLeuOH (1.5 eq) and the DIEA (1.7 eq) in 8 volumes of DCE was added. The mixture was agitated with nitrogen bubbling for 2 hrs. The bed was drained and washed with 1.5 L DCM. The active sites on the resin were end-capped with 1.5 L of a 9:1 MeOH:DIEA solution for 30 minutes. The bed was drained, washed with 4 x 1.5 L of DCM, and dried with a nitrogen purge to give 345 g of loaded resin. Quantitative HPLC analysis was performed by cleaving the Fmoc-amino acid from the resin and assaying versus a standard. HPLC assay of the material showed a loading of the resin at 0.72 mmol/g. Column: Phenomenox Jupiter CIS; 300A; 5^ Flow rate: X mL/min Detection: ITV at 260 nra Mobile phase: A: 0.1% aqueous TFA B: 0.1% TFA in acetonitrile 65% B isocratic Retention time: Approximately 8 minutes 7. EXAHPLE: SOLID PHASE SYNTHESIS OF PEPTIDES Presented below, in Sections 7.l-7.6, are examples of the solid phase synthesis of peptide intermediates as listed in Tables 1, 2, and/or 3. 7.1 Preferred Method for Solid Phase Peptide Synthesis (SPPS); General Procedure A SPPS chamber is charged FmocLeu-resin (1 eq) The resin is conditioned in 5% piperidine DCM (7.5 vol) with a nitrogen purge for 15-30 minutes. The solvent is drained and the resin is treated with 2 x 20% piperidine in NMP (5 volumes) for 30 minutes to remove the Fmoc protecting group. After the second 20% piperidine/NMP treatment, the resin is washed with 5-7 x NMP (5 vol) to a negative choranil test. Meanwhile, the subsequent amino acid {1.5 eq), HOBT {1.5 eq) and DIEA (1.5 eq) are combined in 3:1 NMP/DCM (10 vol), allowed to fully dissolve at room temperature and cooled to O'C. HBTU is added, the solution is stirred for 10-15 minutes to dissolve the solid then added to the resin. The suspension is agitated with stirring under a nitrogen atmosphere for 1-3 hours. Coupling completion is monitored with a qualitative ninhydrin test. If the reaction is incomplete after 3 h (positive ninhydrin test persists) the reactor should be drained and a recoupling should be performed with a fresh solution of activated amino acid (0.5 eq) . Normally after 30 min-1 h of recoupling a negative ninhydrin test is obtained. This cycle is repeated for Che remaining amino acids in the fragment. As the fragment builds, the solvent volumes used in the washes may need to be increased from 5 volumes. In the case of AcAAl-120H end capping with Acetic anhydride was carried out by treating de-Fmoced (HAAl-12-resin) with pyridine (5 eq.) then acetic anhydride (5 eg.) in 3:1 NMP/DCM {10 vol). Following the final coupling, the resin is washed with 3 x 5-6 volumes of NMP then 2 x 10 volumes of DCM and dried to constant weight in a vacuum oven at 4 0°C. 7.2 Preferred Methods for Cleavage of the Peptide from Resin The methods below describe the cleavage of peptide AcAAl-120H from the resin. However, the same methods may be used for cleavage of other peptide fragments of the present invention. Method A: Use of HOAc The resin (l g, 0.370 mmol) was treated with mixture of AcOH / MeOH / DCM (5:1:4, 20 vol, 20 mL) with nitrogen agitation for 1.5 h and the solution was transferred to a round bottom flask, stirred, and treated with water (20 vol). The resulting white slurry was concentrated (rotavap, 40°C bath) to remove DCM and the product collected by filtration. Drying to a constant weight affords 0.69 g (74%) of ACAA1-120H in 87A% purity. A second treatment of the resin as above provided an additional 0,08 g (8.5%) of AcAAi-120H of less pure material (83 Area %) suggesting a desired reaction time of slightly > 1.5 hr. Method B: Use of TFA The resin (1 wt., 20 g) is washed with 5-6 x 1.7 volumes of 1% TFA in DCM, 3-5 minutes each. The 1% TFA/DCM washes are collected in a flask containing pyridine (1:1 volume ratio with the TFA in the wash) . The product containing washes are combined (600 mL, 30 vol) and the DCM is removed by distillation to a minimum pot volume (-1/3 the original volume) . The vacuum is adjusted to maintain a pot temperature of 15-25C. Ethanol (6.5 vol) is added and the distillation is continued until the DCM is removed (as determined by an increase in the temperature of the distillate) . Again the vacuum is adjusted to maintain a pot temperature of 15-20C. The final pot volume should be -8-9 volumes. The solution is cooled to B-ICC and water (6.5 vol) is added over 30 minutes to precipitate the AcAAl-120H. The solid is collected by vacuum filtration and washos with water (2-3 vol). The slurry is stirred at 0-5C for 30 minutes, the solids are collected by vacuum filtration and dried to constant weight to give 16.80 g of AcAAl-120H in 90% yield and 84 Area % (R%) purity. 7.3 Preferred Method for Rework of AGAA1-120H Heat ACAA1-120H (257-21-1, 3.00 g) in 70 mL methanol (23.3 volumes) at 65C with stirring for 3 h. Cool to room terrperature and stir overnight. Suction filtration and drying to constant weight in vacuum oven (40 °C) gave 2.43 g (81%) in 90A%. HPLC Conditions: Vydac 08, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop. 7.4 SPPS of FmocAA13-260H and CleavaQe from the Resin SPPS of PmocAA13-26 was carried out as described above starting with 6.5g of FmocLeuOResin loaded at 1.02 mmol/g. Cleavage method A or B is acceptable (169/137, 60% yield, 85A%). 7 .5 Preferred Rework Procedure for FmocAA13-260H FmocAA13-260H (3.60 g, 85A%) was heated in 15 mL (5 vol) acetonitrile at 78C. The slurry was treated with additional solvent until the solids dissolved (total 0.6 mL). The solution was allowed to cool to room temperature and stirred 7 h. Suction filtration and drying to a constant weight provided 2.6 g (72%) o£ FmocAA13-260H in 95 A%. HPLC Conditions: Vydac CB, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 260 nm. A-. 0.1% TFA / water, B; A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop. 7.6 SPPS of FmocAA27-3S0H and Cleavage from the Reain SPPS of FmocAA27-380H was carried out as described above starting with lOg of FmocTrp(Boc)OR loaded at 0.75 mmol/g. Cleavage method B was used (169/120/1, 78% yield, 87.9A%). HPLC Conditions: Vydac 08, cat. Mo. 20STP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop. 8. EXAMPLE; SOLUTION PHASE SYHTHESIS OF PEPTIDE FRAGMEHTS Presented below, in Sections 8.1 - 8.4, are examples of the solution phase synthesis of peptide intermediates as listed in Tables 1, 2, and/or 3. 8.1 Preparation of Fragment Fmoc-AA27-39-NH2 by-Solution Phase Coupling of HPheMH^ to Fmoc-AA27- 380H FmocAA.2 7 - 3 9NH2 may be prepared by converting the carboxyl terminus of FmocAA27-380H to an activated HOBT or HOAT ester using HBTU or TBTU and HOBT or HOAT, respectively, in the presence of DIEA and phenylalanine amide. The reaction is run in a polar, aprotic solvent such as DMF or NMP at 0 to 25C. At completion of the reaction, alcohol or a water miscible solvent and/or water are added to precipitate FmocAA27-39NH2. Example: FmocAA27-380H (169-120-1, 5.40 g, 1.98 mmol, 1.00 eq.), HPheNHj (Bachem. 0.390 g, 2.38 mmol, 1.20 eq.), HOBT.HjO (0.330 g, 2.16 mmol, 1.10 eq.) were dissolved in NMP (54 mL, 10 vol), treated with DIEA (0.16 mL, 0.921 mmol, 1.10) and stirred at room temperature until dissolved (ca. 30 min) . The solution was cooled using an ice bath (internal T=2-3 °C) and HBTO (0.827 g, 2.18 mmol, 1.10 eq.) was added in one portion, stirred 1 h, then overnight. The reaction can be monitored by TLC (uv, 10% MeOH/DCM, SM mid Rf, product above) . Addition of methanol (54 mL, 10 vol) then water (54 mL, 10 vol) dropwise over a 1 h period formed a free-flowing solid which was stirred an additional 2 h then collected by filtration. The filtercake was washed with 1:1 water/methanol (2 x 25 mL) . Drying overnight in vacuuin oven at 40 **C afforded S.30 g (93%) FmocAA27-39HH2 in 81A%. KPLC analysis also showed that HPLC Conditions: Vydac CS, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min. , 280 nm. A; 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop. 8.2 Preparation of Fragment HAA27-39NH2 by Removal of FMOC Group The Fmoc protecting group of Fmoc AA27-39NHj is removed using a base such as piperidine or potassium carbonate in organic solvents such as DCM, DMF, NMP, methyl C-butyl ether (MTBE), hexane, or mixtures thereof Example: FmocAA2 7-390H {257-25-1, 4.00 g, 1.39 mmol) was slurried in 10 mL (10 vol) of MTBE and 10 mL (2.5 vol) of heptane then treated with piperidine {0.75 mL, 8.34 mmol, 5.5 eq.). The slurry was stirred 12 h at which point 0.5% starting material remained {HPLC) and 20 mL (5 vol) of heptane was added- The free-blowing slurry was stirred 1 h, then isolated by suction filtration and washed 3 x 7 mL (2 vol each washl with 1:1 MTBE / Heptane and dried in a vacuum oven at 40 °C until constant weight affording 3.55 g (96%) of 257-40-1 in 83A% purity. HPLC Conditions: Vydac C8, Cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve l mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop. 8.3 Preparation of Fragment Fnioc-AA13-39-NH2 by Solution-Phase Coupling of Fragments Fmoc-AA13 260H and HAA27-39NH, FmocAA13-39NH2 is prepared by converting the carboxyl terminus of F^ocAA13-260H to an activated HOST or HOAT ester using HETU or TBTU and HOBT or HOAT, respectively, in the presence of DIEA and HAA27-39NH2. The reaction is run in a polar, aprotic solvent such as DMF or NMP at 0 to 25C. At completion of the reaction, alcohol or a water miscible solvent and/or water are added to precipitate FmocAA13-39NH,. Example: FmocAA13-260H (257-41-1, 3.00 g, 0.8417 mrcral, 1 eq.), HAA27-39NH3 (2.23 g, 0.8417 mmol, 1 eq.), HOET hydrate (0.135 g, 0.884 mmol, 1.05 eq.) were dissolved in DMF (2S mL, 30 min.),cooled with an ice bath and treated with DIEA (0.23 tnL, 1.33 mmol, 1.50 eq.) then HBTU (0.335 g, 0.884 mmol, 1.05 eq.) and stirred. After 1 h the reaction was 90% complete based on loss of starting materials (HPLC) and was warmed to room temperature. After 4 h, additional HBTU (0.150 g, 0.395 mmol, 0,5 eq.) was added, and the reaction was stirred overnight. Methanol {50 mL) , then water (50 mL) was added dropwise causing gumming which solidified to a slurry after stirred 16 h at ambient temperature. The solid was-x;ollected by filtration, washed with 2 x 20 mL 1:1 MeOH: water and drying to constant weight giving 5-18 g (99%) of FmocAA13-39NH2 in 60A% purity. The solid 5A% each of acid and amine starting materials. HPLC Conditions: Vydac 08, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. €0-80% B / 30 min-Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop. 8.4 Preparation of Fragment HAA13-39NH2 by Removal of Fmoc Group The FMOC protecting group of FmocAA13-39NH2 is removed using a base such as piperdine or potassium carbonate in. organic solvents such as DCM, DMF, NMP, MTBE, hexane, or mixtures thereof. Example; FmocAA13-390H (257-43-1, 0.500 g, 0.0810 mmol, 1 eg.) was slurried in 9 mL (18 vol) of 2:1MTBE / Heptane for 30 minutes then treated with piperidine (40 μL, 0.404 mmol, 5 eq.) . The slurry was stirred 2 h at which point 40% starting material remained (HPLC) . Additional MTBE (3 mL, 6 vol) and piperidine (20 ^L, 2.5 eq.) were added and the slurry stirred until HPLC Conditions-. Vydac C8, cat. Ko. 208TP5'1, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in O.iO mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop. 9. EXAMPLE! Synthesis of Full Length T-1249 Tseptidea Presented herein, in Sections 9,1 - 9.2, below, are examples of the utilization of the peptide intermediate fragments to produce full length T-1249 peptides. The Example presented in this Section demonstrates the successful coupling of solid phase and solution phase synthesis techniques to produce a full-length T-1249 peptide from peptide intermediate fragments. 9.1 Preparation of Fragment AcAAl-39NH2 by Solution- phase coupling of AcAAl-120H with HAA13-39HH, The synthesis route described here represents the culmination of the T-1249 three fragment approaches schematically depicted in Figure 1. AcAAl-39NH2 may be prepared by converting the carboxyl terminus of AcAAl-120H to an activated HOBT or HOAT ester using HBTU or TBTU and HOBT or HOAT, respectively, in Che presence of DIEA and HAA13-39NH2. At completion of the reaction, alcohol or a water miscible solvent and/or water are added to precipitate AcAAl-SgNH2. Example: HAA13-39 (257-32-2, 0.409 g, 0.0685 mmol, 1 eq.), AcAAl-120H {257-21-1, 0-174 g, 0.0685 mmol, 1 eg.) and HOBT hydrate (0.013 g, 0.0822 mmol, 1.20 eq.) were dissolved in DMF (6.0 mL, 12 vol, 30 min.) . The solution vas cooled with an ice bath and DIEA {18 μL, 0.1028 mmol, 1.5 eq.) followed by HBTU (0.031 g, 0.0822 mmol, 1.20 eq.) . After stirring 4 h at OC, water {6 mL) was added dropwise. The resulting slurry was stirred 1 h then isolated by suction filtration. Drying overnight in a vacuum oven at 40 °C gave 0.545 g (94%) of 257-33-1 in ca. 50A%. HPLC Conditions: Vydac C8, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / rain., 280 nm- A; 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop. 9.2 Preparation of T-X249 Side by Side-chain Deprotection of AcAAl-39NHT The Example presented in this Section demonstrates the successful coupling of solid and liquid phase synthesis techniques to produce a T-1249 peptide from peptide intermediate fragments. In particular, the synthesis route described here represents the final deprotection step of the T-1249 three fragment approach shown in Figure 1. Preferably, the side-chain protecting groups of AcAAl-SPNH2 are removed by acidolysis using 95/5 trifluoroacetic acid/water and up to 5wt/vol% of a carbocation scavenger such as dithiothreitol, ethane dithiol or cystine. The crude T-1249 is precipitated from the deprotection solution by addition of an ether such as MTBE, diethyl ether or diisopropyl ether. Example I ACAAI-39NH3 {257-33-1; 0.120 g) was treated with 1.5 mL of a freshly prepared solution of TFA:DTT:water (95:5:5) and stirred at room temperature for 4 h. MTBE (approximately 3 mL) was added and the precipitate collected by vacuum filtration. The fine powder was dried in the vacuum oven overnight. This material was dissolved in 3 mL of 50% Acetonitrile / water containing 1% HOAc and allowed to stand for 15 hours to allow for decarboxylation of the indole side-chain s"5f the tryptophans. The solution was analyzed directly and found to co-eluted with authentic T'124 9. Approximate purity was 60% by HPLC. HPLC Conditions: Vydac C8, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min. , 280 nm. A: O.I% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B 7 30 min. Typical sample preparation; Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL, into a 20 μl loop. WE CLAIM: !, A method for synthesizing a peptide of the formula: X- WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z {SEQ ID NO: 1), comprising: (a) reacting a side-chain protected peptide of the formula: EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:6), wherein the amino termnimus is deprotected; with a side-chain protected peptide of the formula: X-WQEWEQKITALL-COOH (SEQ ID NO:2) in a known manner, to yield a side-chain protected peptide of the formula: X-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWE'Z (SEQ !D N0:1); wherein X is a protecting group, an acetyl group or a macro molecular carrier group; and wherein Z is a protecting group, or a macromolecular carrier group. 2. The method as claimed in claim 1. wherein X is a protecting group selected from the group consisting of 9-fluoroenylmethoxy-carbonyl (Fmoc). t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), carbobenzoxyl, dansyl and a para-nitrobenzyl ester group. 3. The method as claimed in claim 1, wherein Z is a protecting group selected from the group consisting of 9-fluoroenylmethoxy-carbonyl (Fmoc) t-butyl (t-Bu), trityl (iit), t-butyloxycarbonyl (Boc), carbobenzoxyl, dansyl and a para-nitrobenzyl ester group. 4. The method as claimed in claim 1, wherein X is a macromolecular carrier group selected from the group consisting of lipid-fatly acid conjugates, polyethylene glycol and carbohydrates. 5. The method as claimed in claim 1, wherein Z is a macro molecular carrier group selected from the group consisting of lipid-fatty acid conjugates, polyethylene glycol and carbohydrates. 6. The method as claimed in claim 1, wherein the method includes a step of deprotecting the side chains of the side-chain protected peptide of the formula: X- WQEWEQKITALLEQAQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO:I), 7. The method as claimed in claim 1 or 6, wherein X is an acetyl group. 8. The method as claimed in claim I, wherein Z is an amido group. 9. The method as claimed in claim 1 or 6, wherein X is a protecting group and wherein the method includes a step of modifying X into an acetyl group in a known manner. 10. The method as claimed in claim 9, wherein Z is an amido group. 11. The method as claimed in claim 1, wherein the side-chain protected peptide of the formula: X- WQEWEQKITALL-COOH (SEQ ID N0:2) is synthesized by solid phase peptide synthesis, 12. The method as claimed in claim 4, wherein the side-chain protected peptide of the formula: EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:6) is synthesized by a method comprising: reacting a side-chain protected peptide of the formula: QKLDKWASLWEWF-Z (SEQ ID N0:5), wherein the amino terminus is deprotected; with a side-chain protected peptide of the formula: EQAQIQQEKNEYEL-COOH (SEQ ID N0:3) to yield the side-chain protected peptide of the formula: EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO:6). 13. The method as claimed in 12, wherein the side-chain protected peptide of the formula: QKLDKWASLWEWF-Z (SEQ ID No:5) is synthesized by a method comprising ; reacting a side-chain protected peptide of the formula: QKLDKWASLWEW-COOH (SEQ ID N0:4) with phenylalanine amide to yield the side-chain protected peptide of the formula: QKLDKWASLWEWF-Z (SEQ ID N0.5) 14. The metiiod as claimed in claim 12, wherein the side-chain protected peptide of the formula: EQAQIQQEKNEYEL-COOH (SEQ ID N0:3) is synthesized by solid phase peptide synthesis. 15. The method as claimed in claiml3, wherein the side-chain protected peptide of the formula: QKLDKWASLWEW-COOH (SEQ ID N0:4) is synthesized by solid phase peptide synthesis. 16. A set of peptide intermediate fragments for synthesis of the_peptide as claimed in claim 1, X-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO: 1), selected from the group consisting of: (a) EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 6), WQEWEQKITALL (SEQ ID N0:2); (b) QKLDKWASLWEWF (SEQ ID N0:5), EQAQIQQEKNEYEL (SEQ ID N0:3), WQEWEQKITALL (SEQ ID N0.2); and (c) QKLDKWASLWEW (SEQ ID N0:4), EQAQIQQEKNEYEL (SEQ ID N0:3), WQEWEQKITALL (SEQ ID N0:2). 17. The set of peptide intermediate fragments according to claim 16, wherein one or more of the side chains of said peptide fragments is protected with a protecting group. 18. The set of peptide intermediate fragments according to claim 17, wherein said protecting group is selected from the group consisting of 9-fluoroenylmethoxy-carbpnyl (Fmoc), t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), carbobenzoxyl, dansyl and a para-nitrobenzyl ester group. 19. The set of peptide intermediate fragments as claimed in claim 16, wherein the set comprises: WQEWEQKITALL (SEQ ID N0:2), EQAQIQQEKNEYEL (SEQ ID N0:3), and QKLDKWASLWEW (SEQ ID) N0:4). 20. The set of peptide intermediate fragments as claimed in claim 16, wherein the set comprises: WQEWEQKITALL (SEQ ID N0:2), EQAQIQQEKNEYEL (SEQ ID N0:3), and QKLDKWASLWEWF (SEQ ID N0:5). 21. The set of peptide intermediate fragments as claimed in claim 16, wherein the set comprises: WQEWEQKITALL (SEQ ID N0:2), and EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID N0:6). 22. A peptide selected from the group consisting of: WQEWEQKITALL (SEQ ID N0:2), EQAQIQQEKNEYEL (SEQ ID N0:3) QKLDKWASLWEW (SEQ ID N0:4), QKIDKWASLWEWF (SEQ ID N0:5), EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO:6), WQEWEQKITALLEQAQIQQEKNEYEL (SEQ ID NO. 7), and EQAQIQQEKNEYELQKLDKWASLWEW (SEQ ID NO: 8). 23. A peptide according to claim 22, wherein one or more of the side chains of said peptide is protected with a protecting group. 24. A peptide according to claim 23, wherein said protecting group is selected from the group consisting of 9-fluoroenylmethoxy-carbonyl (Fmoc), t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), carbobenzoxyl dansyl and a para-nitrobenzyl ester group. 25. The peptide as claimed in claim 22, wherein the peptide is WQEWEQKITALL (SEQ ID N0:2). 26. The peptide as claimed in claim 22, wherein the peptide is EQAQIQQEKNEYEL (SEQ ID N0:3). 27. The peptide as claimed in claim 22, wherein the peptide is QKLDKWASLWEW (SEQ ID N0:4). 28. The peptide as claimed in claim 22, wherem the peptide is QKLDKWASLWEWF (SEQ ID NO:5). 29. The peptide as claimed in claim 22, wherein the peptide is EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO:6). 30. Te peptide as claimed in claim 22, wherein the peptide is WQEWEQKITALLEQAQIQQEKNEYEL (SEQ ID N0:7). 31. The peptide as claimed in claim 22, wherein the peptide is EQAQIQQEKNEYELQKLDKWASLWEW (SEQ ID N0:8). 32. A method for synthesizing a peptide substantially as herein described.

Full Text

The present invention relates, first, to methods for the synthesis of peptides, in particular peptides referred to herein as T-1249 (SEQ ID N0:1) and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. The present invention further relates to individual peptide fragments which can act as intermediates in the synthesis of the peptides of interest (e.g.. T-1249). The present invention still further relates to groups of such peptide intermediate fragments which can be utilized together to produce full-length T-1249 and T-1249-like peptides. The present invention still further relates to methods for the purification of peptides, in particular T-1249 and T-1249-like peptides, and the individual peptide fragments which act as intermediates in the synthesis of the subject peptides.
2. BACKGROUND Recently, a large number of peptides have been identified which exhibit an ability to inhibit fusion-associated events, and, importantly, also exhibit potent antiviral activity. See, for exan^le, U.S. Patent Nos. 5,464,933; 5,656,480; PCT Publication Nos. WO 94/28920; WO 96/19495. As these peptides come to be used extensively, for example as therapeutics, the need arises for an ability to synthesize them in large scale quantities.
While techniques exist for peptide synthesis, (see, e.g.. Mergler et al., 1988, Tetrahedron Letters 29:4005-4008; Mergler et al., 1988, Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), "Peptides, Chemistry and Biology, ESCOM, Leiden, 1992, 525-526; and Riniker et al., 1993, Tetrahedron Letters 49:9307-9320) no techniques currently exist which can be utilized for large scale, economical production of easily purified peptides such as T-1249.

3. SUMMARY OF THE INVENTION
The present invention relates, first, to methods for the synthesis of peptides, in particular peptides referred to herein as T-1249 (SEQ ID NO:l) and T-1249-like peptides. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. Generally, the methods of the invention comprise synthesizing specific side-chain protected peptide fragment intermediates of T-1249 or a T-1249-like peptide on a solid support, coupling the protected fragments in solution to form a protected T-1249 or T-1249-like peptide, followed by deprotection of the side chains tc yield the final T-1249 or T-1249-like peptide. A preferred embodiment of the methods of the inver.tion involves the synthesis of a T-1249 peptide having an ami'.no acid sequence as depicted in SHQ ID NO:l.
The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the peptides of interest (e.g., T-1249) . The peptide fragments of the invention include, but are not limited to, those having amino acid sequences as depicted in Table 1 below:

The present invertuion still further relates to particular groups of peptide fragments which act as intermediates in the synthesis of the peptide of interest. The groups of peptide fragments according to the invention include Groups 1-6, as designated in Table 2 below.

This invention is based, in part, on the inventors' unexpected discovery that certain, combinations of solid phase liquid phase methetic reactiorns allow high p-urity T-1249 and T-1249-like peptides to be manufactured for the first time on a large scale with high throughput and high yield. In particular, in accordance with the methods of the invention, T-1249 and T-l249-like peptides may be synthesized on a scale of one or more kilograms. It has been found that by selecting the specific T'1249 peptide fragments of the invention for solid phase synthesis, the highly efficient coupling of solid phase techniques may be exploited without having to use the 3-, 4- or even 5-fold excess of amino acids and reagents that are normally required in solid phase synthesis. The methods of the invention use only about an O.S-fold excess (about 1.5 equivalents) of amino acid in the solid phase synthesis of the peptide fragments of the invention. This reduction in the amount of amino acid and reagents makes the methods of the invention suitable for large scale synthesis of T-1249 and T-1249-like peptides.
In addition, the inventors have surprisingly foxind that certain peptide fragments may be synthesized in the solid phase at a loading of about >o.5 mmol per gram of solid phase resin. This loading significantly enhances throughput over the loading range of 0.25 to 0.35 mmol per gram of resin typically achieved in solid phase peptide synthesis. Moreover, the inventors have found that synthesizing selected peptide fragments in the solid phase using super acid sensitive resin produces peptide fragments of unusually high

purity. Chromatographic techniques are not necessary to purify the peptide fragments produced according to the invention; the fragments are simply put through precipitacion and/or trituration steps before use, or used as obtained directly from the resin. Use of a super acid sensitive resin allows the synthesized, protected peptides of the invention to be cleaved from the resin without concomitant removal of the side-chain protecting groups. This reduces impurities, and allows peptides comprising 10 amino acids or greater to be synthesized in high purity and yield.
The impurity profile of T-1249 and T-1249-like peptides which are synthesized in the solution phase according to the methods of the invention by coupling of the high purity peptide fragments produced according to the invention consists of fragments that did not couple, and contains significantly lower levels of closely related deletion analogues than T-1249 and T-1249-like peptides synthesized according to conventional techniiiues, e.g. . solely solid phase synthesis. Accordingly, T-1249 and T-1249-like peptides produced according to the invention are much easier to purify than those produced according to conventional techniques. In particular, T-1249 produced according to the methods of the present invention may be easily purified to >90% purity in single pass chromatography. For example, in accordance with the methods of the invention, T-1249'can be purified in amounts of 400g or more using a 5 inch column. In contrast, T-1249 prepared using conventional solid phase synthesis The Examples presented in Section 9 below, demonstrate such combinatorial syntheses of T-1249 full-length peptides. The T-1249 and T-1249-like peptides and intermediates may be produced on a scale of one or more kilograms by the methods of the invention.

The presenc inventors have also unexpectedly found that peptides such as T-1249 and other T-l249-like peptides, as well as certain peptide fragments described herein may be purified using high capacity materials because of the high purity of T-1249 after solution phase synthesis. Thus, the present invention still further relates to methods for the purification of peptides, in particular T-1249 and T-1249-like peptides, and the individual peptide fragments which act as intermediates in the synthesis of the subject peptides.
3.1 DEFINITIONS The amino acid notations used herein are conventional
- and are as follows:

4. BRIEF DESCRIPTION OP THE FIGURES
FIG. 1: T-1249 three fragment approach. This figure depicts the scheme followed in the Example presented in Sections 8 and 9 below, for the synthesis of full-length T-1249 beginning with intermediate peptide fragment Group 1, as shown in Table 2, above, and depicts one, non-limiting embodiment of the methods of the invention.

5. DETAILED DESCRIPTION OF THE IHVEHTION 5.1 FULL-LENGTH PEPTIPES The present invention relates to methods, peptide fragments, groups of peptide fragments which can be used to synthesize the peptide known as T-1249. T-1249 is a 39 amino acid residue polypeptide whose sequence is derived from HIV-1, HIV-2 and SIV gp41 viral polypeptide sequences. T-1249 has the following amino acid sequence;
NH2-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-COOH
It will be understood that the methods, fragments and groups of fragments and techniques utilized for choosing the fragments and groups of fragments of the present invention may be used to synthesize T-1249-like fragments in addition to T-1249. The term "T-1249-like" as used herein means any HIV or non-HlV peptide listed in International Publication No. PCT/US99/11212, filed May 20, 1999, which is hereby incorporated by reference in its entirety.
In addition to T'1249 and the T-1249-like peptides described above, the methods, fragments and groups of fragments of the present invention may be used to synthesize peptides having modified amino and/or carboxyl terminal ends. Taking T-1249 as an example, such peptides can be of the formula:
X-WQEWEQKITALLEQAQIQQEKNEyELQKLDKWASLWEWF-Z
wherein X represents an amino group; a hydrophobic group selected from the group consisting of carbobenzoxyl, dansyl, and T-butyloxycarbonyl; an acetyl group; a 9-fluoroenyl-methoxy-carbonyl (FMOC) group; or a macromolecular carrier group selected from the group consisting of lipid-fatty acid conjugates, polyethylene glycol, and carbohydrates; and Z represents a carboxyl group; an amido group; a T-butyloxycarbonyl group; a para-nitrobenzyl ester group; or a macromolecular carrier group selected from the group

consisting of lipid-fatty acid conjugates, polyethylene glycol, and carbohydrates. In a preferred embodiment, the methods of the invention are used to synthesize the peptide having the above formula wherein X is an acetyl group and Z is an amide group. Techniques for addition of such "X" and "Z" groups are well known to those of skill in the art.
In a preferred method, T-1249 and T-1249-like peptides and intermediates can be purified using any non-silica based column packing (for maximization of loading capacity) including but not limited to zirconium-based packings, poly¬styrene, poly-acrylic or other polymer based packings which are stable at high and low pH ranges. For example, among the non-silica-laded column packing exhibiting a broad pH range that includes pH values greater than seven are ones sold by Tosohaas (Montgotneryville, PA) , Columns packed with such material can be run in low, medium or high pressure chromatography according to standard techniques well known in the art.
The Examples presented in Section 9, below, demonstrate the successful synthesis of T-1249 peptides via coupling of peptide intermediates described, below, in Section 5.2.
5.2 PEPTIDE IHTERMEDIATES The present invention encompasses, but is not limited to, peptide fragment intermediates of T-1249 and T-l249-like peptides with specific amino acid sequences as listed in Table 1 above, and the groups of peptide fragment intermediates listed in Table 2. Such peptide intermediates, especially in groups as listed in Table 2, below, can be utilized to produce T-1249 and T-1249 like peptides.
Any one or more of the side-chains of the amino acid residues of peptide fragments listed in Table 1 or 2 may be protected with standard protecting groups such as t-butyl (t-Bu), trityl (trt) and t-butyloxycarbonyl (Boc) . The t-Bu group is the preferred side-chain protecting group for amino acid residues Tyr(Y), Thr{T), Ser(S), Glu(E) and Asp (D) ; the

trt group is the preferred side-chain protecting group for amino acid residues, Gln(Q) and Asn(N); and the Boc group is the preferred side-chain protecting group for amino acid residues Lys(K) and Trp(W).
Preferably, the Gln(Q) residues of the peptide fragments of the invention are protected with trityl (trt) groups. However, if lower solubility of any of the peptide fragments of the invention in organic solvents is desired, the trityl protecting groups may be eliminated from any one or more of the glutamine residues of the fragments.
Preferably, the Asn(N) residues of each peptide fragment of the invention are protected. In addition, it is preferred that the Trp(W) residues are protected with a Boc group.
Protected peptide fragments according to peptide formulas 1-5 listed in Table 1 above include, but are not limited to, the compounds listed in Table 3 below.

Any one or more of the side-chains of the amino acid residues of the peptides listed in Table 3 above may be protected with standard side-chain protecting groups such as tBu, trt and Boc, as described above. Representative synthesis of peptides from Table 3 are presented in Sections 7 and 8 below, which utilize the general techniques discussed in Section 5.3, below.
5.3 PEPTIDE SYNTHESIS As discussed above, some of the individual peptide fragments of the invention are preferably made using solid phase synthesis techniques, while other peptides of the invention are preferably made using a combination of solid phase and solution phase synthesis techniques, said syntheses culminating in the production of T-1249 or T-1249-like peptides as described herein. However, it will be understood that the peptide fragments of the invention/may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, which is incorporated herein by reference in its entirety.
The peptides of the invention may alternatively be synthesized such that one or more of the bonds which link the amino acid residues of the peptides are non-peptide bonds. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds, to name but a few.
In yet another embodiment of the invention, T-1249 and T-1249-like peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the stability, reactivity and/or solubility of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxyl, dansyl, acetyl or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be

placed at the peptides' amino termini. (See "X" modification of T-1249, described above.) Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini. Similarly, a para-nitrobenzyl ester or benzyl ester group may be placed at the peptides' carboxy termini. (See "Z" modification of T-1249, described above.) Technic[ues for introducing such modifications are well known to those of skill in the art.
Further, T-1249 and T-1249-like peptide(s may be synthesized such that their steric configuration as altered. For example, the D-isomer of one or moreof the amino acid residues of the peptide may be used, rather than the usual L-isomer.
Still further, at least one of the amino acid residues of the peptides of the invention may be substituted by one of the well known non-naturally occuring amino acid residues. Alterations such as these may serve to increase the ^ stability, reactivity and/or solubility of the peptides of the invention.
Any of the T-1249 or T-1249-like peptides may be synthesized to additionally have a macromolecular carrier group covalently attached to its amino and/or carboxy termini. Such macromolecular carrier groups may include, for example, lipid-fatty acid conjugates, polyethylene glycol, carbohydrates or additional peptides. The "X" modification of T-1249 described above may therefore additionally represent any of the above macromolecular carrier groups covalently attached to the amino terminus of a peptide, with an additional peptide group being preferred. Likewise, the "Z" modification of T-1249 described above may additionally represent any of the macromolecular carrier groups described above.
Preferably, the peptide fragments of the present invention are synthesized by solid phase peptide synthesis (SPPS) techniques using standard FMOC protocols. See, e.O, . Carpino et al.. 1970, J. Am. Chem. Soc. 92{19}:5748-5749; Carpino et al.. 1972, J. Org. Chem. 37 (22):3404-3409. In a

preferred embodiment, the solid phaae synthesis of the peptide fragments of the present invention is carried out on super acid sensitive solid supports which include, but are not limited to, 2-chlorocritYl chloride resin (see, e.g.. Barlos et al,. 1989, Tetrahedron Letters 30(30):3943-3946} and 4-hydroxymethyl-3-methoxyphenoxybutyric acid resin (see, e.g.. Seiber, 1987, Tetrahedron Letters 28(49):6147-fii50, and Richter et al.. 1994, Tetrahedron Letters 35(27^:4705-4706). Both the 2-chlorotrityl chloride and 4-hydroxymethyl-3-methoxyphenoxy butyric acid resins may be purchased from Calbicchem-Novabiochem Corp., San Diego, CA.
General, non-limiting procedures for production and loading of resins which can be utilized in solid phase peptide synthesis are described herein. In addition, the examples present in Section 6, below, describe exemplary resin preparations.
Resin loading can be performed, for example, via the following techniques: The resin, preferably a super acid sensitive resin such as 2-chloiotrityl resin, is charged to the reaction chamber. The resin is washed with a chlorinated solvent such as dichloromethane (DCM). The bed is drained and a solution of 0.5 - 1,5 equiifalents of an amino acid with an 0.2 to 0.5 excess of diisopropylethylamine (DIEA) in about 8-10 volumes of dichloroethane (DCE) is added. The N-terminus of the amino acid should be protected, preferably with Pmoc, and the side chain of the amino acid should be protected where necessary or appropriate. The mixture is agitated with nitrogen bubbling for 2-24 hours.
It should be noted that a chlorinated solvent such as DCM or DCE is desired for adequate swelling of the 2-chlorotrityl resin.
After agitation, the bed is drained and washed with DCM. The active sites on the resin are endcapped with a 9:1 MeOH:DIEA solution for about 20-30 minutes. The bed is drained, washed four times with DCM and dried with a nitrogen purge to give the loaded resin.

Fmoc is the preferred protecting group for the N-terminus of the amino acid. Depending on which amino acid is being loaded, its side chain may or may not be protected. For example, when tryptophan (Trp) is loaded, its side chain should be protected with Boc. However, it is not necessary to protect the side-chain of leucine (Leu). Preferably, glutamic acid (Glu), aspartic acid (Asp), threonine (Thr) and serine (Ser) are protected as t-butyl ethers or t-butyl esters, and tryptophan (Trp) and lysine (Lys) are protected as t-butoxycarbonyl carbamates (Boc). The amide side-chain of asparagine (Asn) and glutamine (Gin) may or may not be protected with trityl groups.
The Fmoc-protected amino acids used in loading the resin and in peptide synthesis are available, with or without side-chain protecting groups as required, from multiple vendors, including Senn or Genzyme. As an alternative to the above procedure, the resin may be purchased already loaded with the appropriate amino acid.
The Examples presented in Section S, below, describe exemplary resin preparations.
Solid phase peptide synthesis techniques can be performed as, for example, according to the following, non-limiting techniques: The loaded resin is added to the reaction chamber and conditioned with a solvent, preferably methylene chloride (DCM; at preferably about 10 vol.) with nitrogen agitation or stirring for about 15 minutes to swell the resin beads. DCM is required for adequate swelling of the 2-chlorotrityl resin. The resin volume will increase 3-6 fold in the reaction chamber as the beads swell and the active sites vinfold and become accessible to reaction. After tHe resin is swelled, the solvent is drained from the reaction chamber.
Removal of the Fmoc (9-fluroenyl-methyloxycarbonyl) protecting group from the terminal amine or the resin can be accomplished by treating the resin with 2 aliquots of a 20% solution of piperidine in N-methyl-2-pyrrolidinone (NMP) for about ten minutes each. The volume of the 20% solution of

piperidine in NMP required for each aliquot will depend on the scale of the reaction being run. The resin is then washed 5-7 times with aliquots of NMP (aboutio vol.) to remove the Fmoc by-products (i.e., dibenzofulvene and its piperidine adduct) and residual piperidine.
A chloranil test may be used to determine if the removal of residual pyridine is complete. The chloranil test solution is prepared by adding a drop of a saturated solution of chloranil in toluene to about 1 mL of acetone. The NMP washings may be tested by adding a drop of the washing to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine, indicating that residual piperidine is still present. The NMP washing is repeated until the blue or violet color is no longer observed.
Meanwhile, the subsequent amino acid in the sequence to be added to the resin is activated for reaction at its carboxy terminus. The amine terminus of each amino acid should be protected with Fmoc. Depending on which amino acid is being added, its side chain may or may not be protected. Preferably, the side-chains of tyr(y), Thr(T), Ser(S), Glu{E) and Asp(P) are protected with t-Bu, the side-chains of Gln(Q) and Asn(N) are protected with trt, and the side-chains of Lys(K) and Trp{w) are protected with Boc. It is not necessary for the side-chains of Leu or l1e to be protected.
The amino acid can be activated as follows. The Fmoc-protected amino acid (1.5 eq) , 1-hydroxybenzotriazole hydrate (HOBT) (1.5 eq) , and diisopropyl-ethylamine (DIEA) (1.5 eq) are dissolved in a polar, aprotic solvent such as N-methyl pyrrolidinone (NMP), dimethyl formamide (DMF) or dimethyl acetamide (DMAC) (about 7.5 vol.) at room tenperatiire. The solution is chilled to O-S'C, and then O-benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or O-benzotriazol-l-yl-tetramethyltetrafluoroborate (TBTO)(1.5 eq) is added followed by stirring for 5-15 minutes to dissolve. It is important that activation is carried out at O-B'C to minimize racemization of the amino acid. The HBTU is the

last reagent added to the cold solution since activation and racemization cannot take place in its absence.
The solution of activated amino acid is charged to the drained resin, washing in with DCM (approximately 2.5 vol). Note that activation of the amino acid is carried out in NMP due to the insolubility of HBTU in DCM. However, DCM is added to the reaction at this point to maintain adequate swelling of the resin beads. The reaction is agitated with Nj bubbling for about 1 hour at 20-30°C. Coupling completion may be monitored with a qualitative ninhydrin test as described below.
To check for completion of the reaction using the qualitative ninhydrin test, a 2-20 mg sample of the resin can be withdrawn and washed clean with methanol. To the sample is added 3 drops of a 76% solution of phenol in ethanol, 4 or 5 drops of a 0.2 mM KCN solution in pyridine, and 3 drops of a 0.28 M solution of ninhydrin in ethanol. The sample is diluted with ethanol to a volume of about 0.5 mL and placed in a heat block at about 75°C for 5-10 minutes- A blue or violet color is a positive indication for the presence of free amines, indicating that the reaction is not yet complete. The sample can be diluted further to a volume of about 3 mL to more easily gauge the degree of color change in the concentrated sample.
If a positive ninhydrin test is observed after one hour, the coupling reaction is continued for an additional hour. If the positive ninhydrin test persists after 3 hours, the resin is drained, washed one time in approximately 10 volumes of NMP, and the coupling reaction is repeated using 0.5-1 equivalent of activated amino acid.
If the resin is to be stored overnight between coupling cycles, the resin bed may be drained and covered with NMP under a nitrogen blanket. Alternatively, the bed may be drained, stored under a nitrogen blanket, then conditioned with a DCM wash prior to proceeding with the next coupling cycle. If the con^leted fragment is to be stored overnight prior to cleavage, the resin bed should be washed free of NMP

with IPA because significant Fmoc deprotection can occur in NMP.
After the coupling is judged complete, the resin is drained and washed with 3 aliquots (approximately 10 vol.) of NMP. The cycle is repeated for subseguent mers (i.e., amino acids) of the peptide fragment. Following the final coupling reaction, the resin is washed with 4 aliquots (about 10 vol.) of NMP, then with 2 aliquots (approximately 10 vol.) of DCM and 2 IPA. The resin-bound peptide may be dried with a nitrogen purge or in an oven.
Peptides sj-nthesized via solid phase synthesis techniques can be cleaved and isolated according to, for example, the following non-limiting techniques: The peptide may be cleaved from the resin using techniques well known to those skilled in Che art. For example, solutions of 1% or 2% trifluoroacetic acid (TFA) in DCM or a combination of a 1% and a 2% solution of TFA in DCM may be used tc cleave the peptide. Acetic acid (HOAC), hydrochloric acid (HCl) or formic acid may also be used to cleave the peptide. The specific cleavage reagent, solvents and time required for cleavage will depend on the particular peptide being cleaved. After cleavage the cleavage fractions are subjected to standard work-up procedures to isolate the peptide. Typically, the combined cleavage fractions are concentrated under vacuum, followed by reconstitution with polar aprotic or polar aprotic solvents such as ethanol (EtOH), methanol (MeOH), isopropyl alcohol (IPA), acetone, acetonitrile (ACN) , dimethyl formamide (DMF), NMD, DMAC, DCM, etc., followed by precipitation or crystallization with antisolvent such as water or hexanes, and collection by vacuum filtration. Alternatively, the product may be triturated with organic solvents or water after isolation of the peptide.

Accordingly the present invention provides a method for synthesizing a peptide of the formula:
X- WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO: 1), comprising: (a) reacting a side-chain protected peptide of the formula:
EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:6), wherein the amino termninus is deprotected;
with a side-chain protected peptide of the formula:
X-WQE\VEQKITALL-COOH (SEQ ID N0:2) in a known manner, to yield a side-chain protected peptide of the formula:
X-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:1); wherein X is a protecting group, an acetyl group or a macromolccular carrier group; and wherein Z is a protecting group, or a macromolecular carrier group.
The Examples presented in Sections 7.1 - 7.6 below, present solid phase syntheses of peptide intennediates as shown in Tables 1, 2 and/or 3.
For synthesis of full length T-1249 peptides, the peptide intermediates of Table 1, above, can be coupled together to -

yield the T-1249 peptide. For example, the groups of peptide intermediates listed in Table 2, above, can be coupled together to produce T-1249 full-length peptide. Representative examples of the synthesis of full-length T-1249 from intermediate peptide fragments are presented in Section 9, below, and are depicted schematically in Figure 1.
In certain embodiments, a three fragment approach for synthesis of T-1249 can be followed. A "three fragment approach" synthesis refers to a T-1249 synthesis scheme which begins with three T-1249 intermediate peptide fragments that are synthesized and coupled using solid and liquid phase synthesis techniques into a full-length T-1249 peptide. Intermediate peptide fragment groups 1, 2, 3 and 4 shown in Table 2, above, represent preferred groups. Figure 1 depicts an exemplary three fragment approach which utilizes Table 2 peptide intermediate Group 1 to synthesize full-length T-1249. For this group, it is noted that amino acid residue 39 (the T-1249 carboxy 1-terminal amino acid residue) is introduced individually during the fragment coupling process. The culmination of the T-1249 synthesis scheme shown in Figure 1 is demonstrated in the example presented in Section 9.1.
Solution phase peptide synthesis techniques well known to those of skill in the art may be utilized for synthesis of the peptide intermediate fragments of the invention. The Examples presented in Section S describe exemplary solution phase peptide synthesis of peptide intermediates listed in Tables 1, 2, and/or 3. For example, among the non-silica-laded column packing exhibiting a broad pH range that includes pH stability values at high or low pH are sold by Tosohaas (Montgomeryville, PA).

The air sensitive 2-chlorotritYlchloride resin (Senn Chemicals, Lot A 3573, 1 eq, 12.0 inmol, 10.0 g) is added to a 250 mL round bottom flask and immediately treated with a prepared solution of FmocLeuOH (1.0 eq. , 12 mmol, 4.24 g) and Diisopropyl ethyl amine (5eq., 60.0 mmol, 5.20 mL) in DCN {10 vol, 100 mL) The slurry is capped and stirred for 3 hours. The solvent is removed by filtration and the resin is v.'ashed with DCM (5 vol, 50 mL) . The remaining active sites on the resin are end-capped by treating resin with 9:1 MeOH:DiEA (5 vol, 5.0 mL DJEA and 45 mL MeOH) for 30 minutes. The solvent is removed and the resin is washed with 3x5 volumes of DCM. The resin is dried to constant weight affording 13.2 g of loaded resin with a calculated loading of 0.98 mmol FmocLeuOH per gram.
This Method can also be used for Loading FmocTrp (Boc)OH onto 2-CTC resin.
Described herein, in Sections 6.2-6.3, are examples in which chlorotrityl chloride resins were substituted with amino acids which can be utilized in conjunction with solid phase synthesis of the peptides and peptide intermediates-described herein. All of the peptides and peptide fragments of the present invention may be synthesized by solid phase peptide synthesis (EPPS) using the loading procedures described in Sections 6.2 and 6.3 below.

Procedure:
The 2-chlorotrityl chloride resin (25 g, 1 eq.) was charged to a 500 mL peptide chamber and washed with 250 mL of DCM. The bed was drained and a solution of the Fmoc-Trp(Boc)-OH {1.5 eq) and the DIEA (1.7 eq) in 10 volumes of DCE was added. The mixture was agitated with nitrogen bubbling for 2 hrs.
The bed was drained and washed with 250 mL DCM- The active sites on the resin were end-capped with 200 mL of a 9:i MeOHiDIEA solution for 20 minutes. The bed was drained, v/ashed with 4 x 250 rnL of DCM, and dried with a nitrogen purge to give 34.3 g of loaded resin.
Quantitative HPLC analysis was performed by cleaving the Fmoc-amino acid from the resin and assaying versus a standard.
HPLC assay of the material showed a loading of the resin at 0.68 mmol/g.
Column: Phenomenox Jupiter C18; 300A; 5μ Plow rate: 1 mL/min Detection: UV at 260 nm
Mobile phase: A: 0.1% aqueous TFA
B: 0.1% TFA in acetonitrile 65% B isocratic
Retention time: Approximately 14 minutes

Procedure{
The resin was charged to a 3 L peptide chamber and washed with 1.5 DCM. The bed was drained and a solution of the FmocLeuOH (1.5 eq) and the DIEA (1.7 eq) in 8 volumes of DCE was added. The mixture was agitated with nitrogen bubbling for 2 hrs.

The bed was drained and washed with 1.5 L DCM. The active sites on the resin were end-capped with 1.5 L of a 9:1 MeOH:DIEA solution for 30 minutes. The bed was drained, washed with 4 x 1.5 L of DCM, and dried with a nitrogen purge to give 345 g of loaded resin.
Quantitative HPLC analysis was performed by cleaving the
Fmoc-amino acid from the resin and assaying versus a
standard. HPLC assay of the material showed a loading of
the resin at 0.72 mmol/g.
Column: Phenomenox Jupiter CIS; 300A; 5^
Flow rate: X mL/min
Detection: ITV at 260 nra
Mobile phase: A: 0.1% aqueous TFA
B: 0.1% TFA in acetonitrile
65% B isocratic
Retention time: Approximately 8 minutes
7. EXAHPLE: SOLID PHASE SYNTHESIS OF PEPTIDES
Presented below, in Sections 7.l-7.6, are examples of the solid phase synthesis of peptide intermediates as listed in Tables 1, 2, and/or 3.
7.1 Preferred Method for Solid Phase Peptide Synthesis
(SPPS); General Procedure
A SPPS chamber is charged FmocLeu-resin (1 eq) The resin is conditioned in 5% piperidine DCM (7.5 vol) with a nitrogen purge for 15-30 minutes. The solvent is drained and the resin is treated with 2 x 20% piperidine in NMP (5 volumes) for 30 minutes to remove the Fmoc protecting group. After the second 20% piperidine/NMP treatment, the resin is washed with 5-7 x NMP (5 vol) to a negative choranil test.
Meanwhile, the subsequent amino acid {1.5 eq), HOBT {1.5 eq) and DIEA (1.5 eq) are combined in 3:1 NMP/DCM (10 vol), allowed to fully dissolve at room temperature and cooled to O'C. HBTU is added, the solution is stirred for 10-15 minutes to dissolve the solid then added to the resin. The suspension is agitated with stirring under a nitrogen

atmosphere for 1-3 hours. Coupling completion is monitored with a qualitative ninhydrin test. If the reaction is incomplete after 3 h (positive ninhydrin test persists) the reactor should be drained and a recoupling should be performed with a fresh solution of activated amino acid (0.5 eq) . Normally after 30 min-1 h of recoupling a negative ninhydrin test is obtained. This cycle is repeated for Che remaining amino acids in the fragment. As the fragment builds, the solvent volumes used in the washes may need to be increased from 5 volumes. In the case of AcAAl-120H end capping with Acetic anhydride was carried out by treating de-Fmoced (HAAl-12-resin) with pyridine (5 eq.) then acetic anhydride (5 eg.) in 3:1 NMP/DCM {10 vol). Following the final coupling, the resin is washed with 3 x 5-6 volumes of NMP then 2 x 10 volumes of DCM and dried to constant weight in a vacuum oven at 4 0°C.
7.2 Preferred Methods for Cleavage of the Peptide from
Resin
The methods below describe the cleavage of peptide AcAAl-120H from the resin. However, the same methods may be used for cleavage of other peptide fragments of the present invention.
Method A: Use of HOAc
The resin (l g, 0.370 mmol) was treated with mixture of AcOH / MeOH / DCM (5:1:4, 20 vol, 20 mL) with nitrogen agitation for 1.5 h and the solution was transferred to a round bottom flask, stirred, and treated with water (20 vol). The resulting white slurry was concentrated (rotavap, 40°C bath) to remove DCM and the product collected by filtration. Drying to a constant weight affords 0.69 g (74%) of ACAA1-120H in 87A% purity. A second treatment of the resin as above provided an additional 0,08 g (8.5%) of AcAAi-120H of less pure material (83 Area %) suggesting a desired reaction time of slightly > 1.5 hr.

Method B: Use of TFA
The resin (1 wt., 20 g) is washed with 5-6 x 1.7 volumes of 1% TFA in DCM, 3-5 minutes each. The 1% TFA/DCM washes are collected in a flask containing pyridine (1:1 volume ratio with the TFA in the wash) . The product containing washes are combined (600 mL, 30 vol) and the DCM is removed by distillation to a minimum pot volume (-1/3 the original volume) . The vacuum is adjusted to maintain a pot temperature of 15-25C. Ethanol (6.5 vol) is added and the distillation is continued until the DCM is removed (as determined by an increase in the temperature of the distillate) . Again the vacuum is adjusted to maintain a pot temperature of 15-20C. The final pot volume should be -8-9 volumes. The solution is cooled to B-ICC and water (6.5 vol) is added over 30 minutes to precipitate the AcAAl-120H. The solid is collected by vacuum filtration and washos with water (2-3 vol). The slurry is stirred at 0-5C for 30 minutes, the solids are collected by vacuum filtration and dried to constant weight to give 16.80 g of AcAAl-120H in 90% yield and 84 Area % (R%) purity.
7.3 Preferred Method for Rework of AGAA1-120H
Heat ACAA1-120H (257-21-1, 3.00 g) in 70 mL methanol (23.3 volumes) at 65C with stirring for 3 h. Cool to room terrperature and stir overnight. Suction filtration and drying to constant weight in vacuum oven (40 °C) gave 2.43 g (81%) in 90A%.
HPLC Conditions: Vydac 08, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop.

7.4 SPPS of FmocAA13-260H and CleavaQe from the Resin
SPPS of PmocAA13-26 was carried out as described above starting with 6.5g of FmocLeuOResin loaded at 1.02 mmol/g. Cleavage method A or B is acceptable (169/137, 60% yield, 85A%).
7 .5 Preferred Rework Procedure for FmocAA13-260H
FmocAA13-260H (3.60 g, 85A%) was heated in 15 mL (5 vol) acetonitrile at 78C. The slurry was treated with additional solvent until the solids dissolved (total 0.6 mL). The solution was allowed to cool to room temperature and stirred 7 h. Suction filtration and drying to a constant weight provided 2.6 g (72%) o£ FmocAA13-260H in 95 A%.
HPLC Conditions: Vydac CB, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 260 nm. A-. 0.1% TFA / water, B; A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop.
7.6 SPPS of FmocAA27-3S0H and Cleavage from the Reain
SPPS of FmocAA27-380H was carried out as described above starting with lOg of FmocTrp(Boc)OR loaded at 0.75 mmol/g. Cleavage method B was used (169/120/1, 78% yield, 87.9A%).
HPLC Conditions: Vydac 08, cat. Mo. 20STP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop.

8. EXAMPLE; SOLUTION PHASE SYHTHESIS OF PEPTIDE FRAGMEHTS
Presented below, in Sections 8.1 - 8.4, are examples of the solution phase synthesis of peptide intermediates as listed in Tables 1, 2, and/or 3.
8.1 Preparation of Fragment Fmoc-AA27-39-NH2 by-Solution Phase Coupling of HPheMH^ to Fmoc-AA27-
380H
FmocAA.2 7 - 3 9NH2 may be prepared by converting the
carboxyl terminus of FmocAA27-380H to an activated HOBT or
HOAT ester using HBTU or TBTU and HOBT or HOAT, respectively,
in the presence of DIEA and phenylalanine amide. The
reaction is run in a polar, aprotic solvent such as DMF or
NMP at 0 to 25C. At completion of the reaction, alcohol or a
water miscible solvent and/or water are added to precipitate
FmocAA27-39NH2.
Example:
FmocAA27-380H (169-120-1, 5.40 g, 1.98 mmol, 1.00 eq.), HPheNHj (Bachem. 0.390 g, 2.38 mmol, 1.20 eq.), HOBT.HjO (0.330 g, 2.16 mmol, 1.10 eq.) were dissolved in NMP (54 mL, 10 vol), treated with DIEA (0.16 mL, 0.921 mmol, 1.10) and stirred at room temperature until dissolved (ca. 30 min) . The solution was cooled using an ice bath (internal T=2-3 °C) and HBTO (0.827 g, 2.18 mmol, 1.10 eq.) was added in one portion, stirred 1 h, then overnight. The reaction can be monitored by TLC (uv, 10% MeOH/DCM, SM mid Rf, product above) . Addition of methanol (54 mL, 10 vol) then water (54 mL, 10 vol) dropwise over a 1 h period formed a free-flowing solid which was stirred an additional 2 h then collected by filtration. The filtercake was washed with 1:1 water/methanol (2 x 25 mL) . Drying overnight in vacuuin oven at 40 **C afforded S.30 g (93%) FmocAA27-39HH2 in 81A%. KPLC analysis also showed that
HPLC Conditions: Vydac CS, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min. , 280 nm. A; 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop.
8.2 Preparation of Fragment HAA27-39NH2 by Removal of
FMOC Group
The Fmoc protecting group of Fmoc AA27-39NHj is removed using a base such as piperidine or potassium carbonate in organic solvents such as DCM, DMF, NMP, methyl C-butyl ether (MTBE), hexane, or mixtures thereof
Example:
FmocAA2 7-390H {257-25-1, 4.00 g, 1.39 mmol) was slurried in 10 mL (10 vol) of MTBE and 10 mL (2.5 vol) of heptane then treated with piperidine {0.75 mL, 8.34 mmol, 5.5 eq.). The slurry was stirred 12 h at which point 0.5% starting material remained {HPLC) and 20 mL (5 vol) of heptane was added- The free-blowing slurry was stirred 1 h, then isolated by suction filtration and washed 3 x 7 mL (2 vol each washl with 1:1 MTBE / Heptane and dried in a vacuum oven at 40 °C until constant weight affording 3.55 g (96%) of 257-40-1 in 83A% purity.
HPLC Conditions: Vydac C8, Cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve l mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop.

8.3 Preparation of Fragment Fnioc-AA13-39-NH2 by
Solution-Phase Coupling of Fragments Fmoc-AA13
260H and HAA27-39NH,
FmocAA13-39NH2 is prepared by converting the carboxyl terminus of F^ocAA13-260H to an activated HOST or HOAT ester using HETU or TBTU and HOBT or HOAT, respectively, in the presence of DIEA and HAA27-39NH2. The reaction is run in a polar, aprotic solvent such as DMF or NMP at 0 to 25C. At completion of the reaction, alcohol or a water miscible solvent and/or water are added to precipitate FmocAA13-39NH,.
Example:
FmocAA13-260H (257-41-1, 3.00 g, 0.8417 mrcral, 1 eq.), HAA27-39NH3 (2.23 g, 0.8417 mmol, 1 eq.), HOET hydrate (0.135 g, 0.884 mmol, 1.05 eq.) were dissolved in DMF (2S mL, 30 min.),cooled with an ice bath and treated with DIEA (0.23 tnL, 1.33 mmol, 1.50 eq.) then HBTU (0.335 g, 0.884 mmol, 1.05 eq.) and stirred. After 1 h the reaction was 90% complete based on loss of starting materials (HPLC) and was warmed to room temperature. After 4 h, additional HBTU (0.150 g, 0.395 mmol, 0,5 eq.) was added, and the reaction was stirred overnight. Methanol {50 mL) , then water (50 mL) was added dropwise causing gumming which solidified to a slurry after stirred 16 h at ambient temperature. The solid was-x;ollected by filtration, washed with 2 x 20 mL 1:1 MeOH: water and drying to constant weight giving 5-18 g (99%) of FmocAA13-39NH2 in 60A% purity. The solid 5A% each of acid and amine starting materials.
HPLC Conditions: Vydac 08, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. €0-80% B / 30 min-Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 uL into a 20 uL loop.

8.4 Preparation of Fragment HAA13-39NH2 by Removal of
Fmoc Group
The FMOC protecting group of FmocAA13-39NH2 is removed using a base such as piperdine or potassium carbonate in. organic solvents such as DCM, DMF, NMP, MTBE, hexane, or mixtures thereof.
Example;
FmocAA13-390H (257-43-1, 0.500 g, 0.0810 mmol, 1 eg.) was slurried in 9 mL (18 vol) of 2:1MTBE / Heptane for 30 minutes then treated with piperidine (40 μL, 0.404 mmol, 5 eq.) . The slurry was stirred 2 h at which point 40% starting material remained (HPLC) . Additional MTBE (3 mL, 6 vol) and piperidine (20 ^L, 2.5 eq.) were added and the slurry stirred until HPLC Conditions-. Vydac C8, cat. Ko. 208TP5'1, 5 u, 300 A, 0.9 mL / min., 280 nm. A: 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in O.iO mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop.
9. EXAMPLE! Synthesis of Full Length T-1249 Tseptidea Presented herein, in Sections 9,1 - 9.2, below, are examples of the utilization of the peptide intermediate fragments to produce full length T-1249 peptides.
The Example presented in this Section demonstrates the successful coupling of solid phase and solution phase synthesis techniques to produce a full-length T-1249 peptide from peptide intermediate fragments.

9.1 Preparation of Fragment AcAAl-39NH2 by Solution-
phase coupling of AcAAl-120H with HAA13-39HH,
The synthesis route described here represents the culmination of the T-1249 three fragment approaches schematically depicted in Figure 1. AcAAl-39NH2 may be prepared by converting the carboxyl terminus of AcAAl-120H to an activated HOBT or HOAT ester using HBTU or TBTU and HOBT or HOAT, respectively, in Che presence of DIEA and HAA13-39NH2. At completion of the reaction, alcohol or a water miscible solvent and/or water are added to precipitate AcAAl-SgNH2.
Example:
HAA13-39 (257-32-2, 0.409 g, 0.0685 mmol, 1 eq.), AcAAl-120H {257-21-1, 0-174 g, 0.0685 mmol, 1 eg.) and HOBT hydrate
(0.013 g, 0.0822 mmol, 1.20 eq.) were dissolved in DMF (6.0 mL, 12 vol, 30 min.) . The solution vas cooled with an ice bath and DIEA {18 μL, 0.1028 mmol, 1.5 eq.) followed by HBTU
(0.031 g, 0.0822 mmol, 1.20 eq.) . After stirring 4 h at OC, water {6 mL) was added dropwise. The resulting slurry was stirred 1 h then isolated by suction filtration. Drying overnight in a vacuum oven at 40 °C gave 0.545 g (94%) of 257-33-1 in ca. 50A%.
HPLC Conditions: Vydac C8, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / rain., 280 nm- A; 0.1% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B / 30 min. Typical sample preparation: Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL into a 20 μL loop.
9.2 Preparation of T-X249 Side by Side-chain
Deprotection of AcAAl-39NHT
The Example presented in this Section demonstrates the successful coupling of solid and liquid phase synthesis techniques to produce a T-1249 peptide from peptide intermediate fragments. In particular, the synthesis route

described here represents the final deprotection step of the T-1249 three fragment approach shown in Figure 1. Preferably, the side-chain protecting groups of AcAAl-SPNH2 are removed by acidolysis using 95/5 trifluoroacetic acid/water and up to 5wt/vol% of a carbocation scavenger such as dithiothreitol, ethane dithiol or cystine. The crude T-1249 is precipitated from the deprotection solution by addition of an ether such as MTBE, diethyl ether or diisopropyl ether.
Example I
ACAAI-39NH3 {257-33-1; 0.120 g) was treated with 1.5 mL of a freshly prepared solution of TFA:DTT:water (95:5:5) and stirred at room temperature for 4 h. MTBE (approximately 3 mL) was added and the precipitate collected by vacuum filtration. The fine powder was dried in the vacuum oven overnight. This material was dissolved in 3 mL of 50% Acetonitrile / water containing 1% HOAc and allowed to stand for 15 hours to allow for decarboxylation of the indole side-chain s"5f the tryptophans. The solution was analyzed directly and found to co-eluted with authentic T'124 9. Approximate purity was 60% by HPLC.
HPLC Conditions: Vydac C8, cat. No. 208TP54, 5 u, 300 A, 0.9 mL / min. , 280 nm. A: O.I% TFA / water, B: A mixture of 80% I-PrOH / 20% Acetonitrile and 0.1% TFA. 60-80% B 7 30 min. Typical sample preparation; Dissolve 1 mg in 0.10 mL NMP, dilute with 1 mL Acetonitrile. Inject 20 μL, into a 20 μl loop.

WE CLAIM:
!, A method for synthesizing a peptide of the formula:
X- WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z {SEQ ID NO:
1), comprising:
(a) reacting a side-chain protected peptide of the formula:
EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:6), wherein the amino termnimus is deprotected;
with a side-chain protected peptide of the formula:
X-WQEWEQKITALL-COOH (SEQ ID NO:2) in a known manner, to yield a side-chain protected peptide of the formula:
X-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWE'Z (SEQ !D N0:1); wherein X is a protecting group, an acetyl group or a macro molecular carrier group; and wherein Z is a protecting group, or a macromolecular carrier group.
2. The method as claimed in claim 1. wherein X is a protecting group selected from the group consisting of 9-fluoroenylmethoxy-carbonyl (Fmoc). t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), carbobenzoxyl, dansyl and a para-nitrobenzyl ester group.
3. The method as claimed in claim 1, wherein Z is a protecting group selected from the group consisting of 9-fluoroenylmethoxy-carbonyl (Fmoc) t-butyl (t-Bu), trityl (iit), t-butyloxycarbonyl (Boc), carbobenzoxyl, dansyl and a para-nitrobenzyl ester group.
4. The method as claimed in claim 1, wherein X is a macromolecular carrier group selected from the group consisting of lipid-fatly acid conjugates, polyethylene glycol and carbohydrates.

5. The method as claimed in claim 1, wherein Z is a macro molecular carrier group selected from the group consisting of lipid-fatty acid conjugates, polyethylene glycol and carbohydrates.
6. The method as claimed in claim 1, wherein the method includes a step of deprotecting the side chains of the side-chain protected peptide of the formula:
X- WQEWEQKITALLEQAQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO:I),
7. The method as claimed in claim 1 or 6, wherein X is an acetyl group.
8. The method as claimed in claim I, wherein Z is an amido group.
9. The method as claimed in claim 1 or 6, wherein X is a protecting group and wherein the method includes a step of modifying X into an acetyl group in a known manner.
10. The method as claimed in claim 9, wherein Z is an amido group.
11. The method as claimed in claim 1, wherein the side-chain protected peptide of the formula:
X- WQEWEQKITALL-COOH (SEQ ID N0:2) is synthesized by solid phase peptide synthesis,
12. The method as claimed in claim 4, wherein the side-chain protected peptide of
the formula:
EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID N0:6) is synthesized by a
method comprising:
reacting a side-chain protected peptide of the formula:
QKLDKWASLWEWF-Z (SEQ ID N0:5), wherein the amino terminus is deprotected;
with a side-chain protected peptide of the formula:
EQAQIQQEKNEYEL-COOH (SEQ ID N0:3)
to yield the side-chain protected peptide of the formula:
EQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO:6).

13. The method as claimed in 12, wherein the side-chain protected peptide of the
formula: QKLDKWASLWEWF-Z (SEQ ID No:5) is synthesized by a method
comprising ;
reacting a side-chain protected peptide of the formula:
QKLDKWASLWEW-COOH (SEQ ID N0:4)
with phenylalanine amide to yield the side-chain protected peptide of the
formula:
QKLDKWASLWEWF-Z (SEQ ID N0.5)
14. The metiiod as claimed in claim 12, wherein the side-chain protected peptide of
the formula:
EQAQIQQEKNEYEL-COOH (SEQ ID N0:3) is synthesized by solid phase peptide synthesis.
15. The method as claimed in claiml3, wherein the side-chain protected peptide of
the formula:
QKLDKWASLWEW-COOH (SEQ ID N0:4) is synthesized by solid phase peptide synthesis.
16. A set of peptide intermediate fragments for synthesis of the_peptide as claimed
in claim 1,
X-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-Z (SEQ ID NO: 1), selected from the group consisting of:
(a) EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 6), WQEWEQKITALL (SEQ ID N0:2);
(b) QKLDKWASLWEWF (SEQ ID N0:5), EQAQIQQEKNEYEL (SEQ ID N0:3), WQEWEQKITALL (SEQ ID N0.2); and

(c) QKLDKWASLWEW (SEQ ID N0:4), EQAQIQQEKNEYEL (SEQ ID N0:3), WQEWEQKITALL (SEQ ID N0:2).
17. The set of peptide intermediate fragments according to claim 16, wherein one or more of the side chains of said peptide fragments is protected with a protecting group.
18. The set of peptide intermediate fragments according to claim 17, wherein said protecting group is selected from the group consisting of 9-fluoroenylmethoxy-carbpnyl (Fmoc), t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), carbobenzoxyl, dansyl and a para-nitrobenzyl ester group.
19. The set of peptide intermediate fragments as claimed in claim 16, wherein the set comprises:
WQEWEQKITALL (SEQ ID N0:2), EQAQIQQEKNEYEL (SEQ ID N0:3), and QKLDKWASLWEW (SEQ ID) N0:4).
20. The set of peptide intermediate fragments as claimed in claim 16, wherein the
set comprises:
WQEWEQKITALL (SEQ ID N0:2), EQAQIQQEKNEYEL (SEQ ID N0:3), and QKLDKWASLWEWF (SEQ ID N0:5).
21. The set of peptide intermediate fragments as claimed in claim 16, wherein the
set comprises:
WQEWEQKITALL (SEQ ID N0:2), and EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID N0:6).

22. A peptide selected from the group consisting of: WQEWEQKITALL (SEQ ID N0:2), EQAQIQQEKNEYEL (SEQ ID N0:3) QKLDKWASLWEW (SEQ ID N0:4), QKIDKWASLWEWF (SEQ ID N0:5), EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO:6), WQEWEQKITALLEQAQIQQEKNEYEL (SEQ ID NO. 7), and EQAQIQQEKNEYELQKLDKWASLWEW (SEQ ID NO: 8).
23. A peptide according to claim 22, wherein one or more of the side chains of said peptide is protected with a protecting group.
24. A peptide according to claim 23, wherein said protecting group is selected from the group consisting of 9-fluoroenylmethoxy-carbonyl (Fmoc), t-butyl (t-Bu), trityl (trt), t-butyloxycarbonyl (Boc), carbobenzoxyl dansyl and a para-nitrobenzyl ester group.
25. The peptide as claimed in claim 22, wherein the peptide is WQEWEQKITALL (SEQ ID N0:2).
26. The peptide as claimed in claim 22, wherein the peptide is EQAQIQQEKNEYEL (SEQ ID N0:3).

27. The peptide as claimed in claim 22, wherein the peptide is QKLDKWASLWEW (SEQ ID N0:4).
28. The peptide as claimed in claim 22, wherem the peptide is QKLDKWASLWEWF (SEQ ID NO:5).

29. The peptide as claimed in claim 22, wherein the peptide is
EQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO:6).
30. Te peptide as claimed in claim 22, wherein the peptide is WQEWEQKITALLEQAQIQQEKNEYEL (SEQ ID N0:7).
31. The peptide as claimed in claim 22, wherein the peptide is EQAQIQQEKNEYELQKLDKWASLWEW (SEQ ID N0:8).
32. A method for synthesizing a peptide substantially as herein described.

Documents:

in-pct-2002-154-che abstract.pdf

in-pct-2002-154-che claims-duplicate.pdf

in-pct-2002-154-che claims.pdf

in-pct-2002-154-che correspondence-others.pdf

in-pct-2002-154-che correspondence-po.pdf

in-pct-2002-154-che description (complete)-duplicate.pdf

in-pct-2002-154-che description (complete).pdf

in-pct-2002-154-che drawings.pdf

in-pct-2002-154-che form-1.pdf

in-pct-2002-154-che form-26.pdf

in-pct-2002-154-che form-3.pdf

in-pct-2002-154-che form-5.pdf

in-pct-2002-154-che others.pdf

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Patent Number

235200

Indian Patent Application Number

IN/PCT/2002/154/CHE

PG Journal Number

29/2009

Publication Date

17-Jul-2009

Grant Date

25-Jun-2009

Date of Filing

28-Jan-2002

Name of Patentee

TRIMERIS, INC.

Applicant Address

4727 University Drive, Durham, NC 27707

Inventors:

#	Inventor's Name	Inventor's Address
1	BRAY, Brian	1912 Broadway Drive Graham, NC 27253
2	ANDERSEN, Marc	4108 Balsam Drive Raleigh, NC 27612
3	FRIEDRICH, Paul, E.	4208 Wyckford Place Apex, NC 27502
4	KANG, Myung-Chol	193 Landing Drive Chapel Hill, NC 27514

PCT International Classification Number

C07K1/00

PCT International Application Number

PCT/US00/35725

PCT International Filing date

2000-07-05

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	09/349,205	1999-07-07	U.S.A.